The aim of the proposed research is to further our understanding of the role of the platelet membrane skeleton in regulating platelet function. In recent work, we have shown that platelets contain a membrane skeleton that appears to be a distinct component of the platelet cytoskeleton. We have suggested that this skeleton maintains the platelet in a functional form and that activation-induced changes to the skeleton may be responsible for some platelet responses. Actin-binding protein is a major component and links the skeleton to the glycoprotein (GP) Ib-IX complex on the plasma membrane. Additional proteins that co-isolate with the membrane skeleton are spectrin and GP Ia-IIa. The aims of the present proposal are: 1) to characterize the organization of the platelet membrane skeleton; 2) to characterize the interaction between actin-binding protein and the GP Ib-IX complex; 3) to identify platelet functions regulated by the membrane skeleton; 4) to test the hypothesis that the membrane skeleton is reorganized when calpain is activated in platelets. The proteins present in the membrane skeleton will be identified by isolating the skeletons and analyzing their composition on gels and immunoblots. Association of proteins with the skeleton will be confirmed morphologically. The organization of individual components will be determined by high-resolution electron microscopy of the membrane skeleton. Linkage mechanisms between the skeleton and glycoproteins such as the GP Ib-IX complex and GP Ia-IIa will be characterized. The GP Ib-IX complex has been expressed in cultured cells; it associated with the cytoskeleton. Thus, functional domains on this complex will be identified by expression of native or truncated forms. The domain on actin-binding protein that is involved in the association with the GP Ib-IX complex will be identified. Truncated or mutated forms of actin-binding protein will be made by expression in a bacterial system. Antibodies against the functional domain of actin-binding protein will be injected into cultured cells to test the hypothesis that actin-binding protein mediates the association of the cytoskeleton with the membrane of cells other than the platelet. The hypothesis that the association of the GP Ib-IX complex with the membrane skeleton regulates the lateral distribution and ligand-binding activity of the GP Ib-IX complex will be examined by expression of native or truncated forms of GP Ib-IX. Finally, by comparing the structure of the membrane skeleton in unstimulated platelets with that in platelets in which calpain has been activated, the hypothesis that calpain induces functional changes in activated platelets as a result of reorganization of the membrane skeleton will be tested. These experiments should provide a detailed understanding of the organization of the platelet membrane skeleton and the role of this structure in the regulation of platelet function.